Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa
Basic protein information
Accession P14907
Common name Nsp1, Nsp1p
Gene NSP1
Organism Saccharomyces cerevisiae
Uniprot name Nucleoporin NSP1
Basic LLPS information
Organelle nuclear pore central transport channel; selective hydrogel-like meshwork formed by FG-nucleoporins in nuclear pore central channel
Type of experimental evidence
Protein region(s) mediating LLPS
2
-
601
FG-rich repeats with N-rich inter-FG spacers
Based on the experimental results of the following publication: 20304795
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
The permeability barrier of nuclear pore complexes (NPCs) controls the exchange between nucleus and cytoplasm. It suppresses the flux of inert macromolecules >30 kDa but allows rapid passage of even very large cargoes, provided these are bound to appropriate nuclear transport receptors (facilitated translocation). FG-rich nucleoporin repeats constitute the permeability barrier, they are essential for viability and engage in two known kinds of interactions: binding of NTRs and hydrogel formation that arises through inter-repeat contact. The F→S mutated Nsp1 repeats failed to form a hydrogel (PMID:17082456). A saturated hydrogel formed by a single nucleoporin FG-repeat domain is sufficient to reproduce the permeability properties of NPCs. Importin beta and related nuclear transport receptors entered such hydrogel >1000x faster than a similarly sized inert macromolecule (PMID:17693259). The NQTS-rich sequences of nucleoporins connect FG motifs in a repeat domain. In contrast to previous belief, they are, however, not just functionless spacers. Instead, they engage in amyloid-like protein-protein contacts that presumably tighten the FG hydrogel-based permeability barrier of NPCs. The cohesiveness of the NQTS-rich FG repeats appears to be so akin to that of the NQ-rich prion domain of Sup35p that the two modules interact with each other. However, while NQ-rich amyloids are very dense, tightly packed structures, where side chain stacking generates an additional anhydrous peptide interface between the β-sheets, FG hydrogel formation apparently stops before a complete collapse of the structure. Consequently, FG hydrogels include water and allow passive entry of small molecules and facilitated entry of NTRs. Too strong inter-FG repeat interactions might be counteracted by the presence of residues that form weaker β-sheets than Gln, such as Ser or Gly. Likewise, while the N-terminal segment of Nsp1p (residues 2-277) alone forms more strong hydrogels, presence of the charged C-terminal FxFG repeats of Nsp1p (residues 274-601) apparently modulate the gel strength such that NTRs can move ≈3-fold faster through the gel. Therefore, in contrast to pathological amyloids, inter-FG repeat contacts do not result in irreversible aggregates (PMID:20304795).
Literature supporting the LLPS: 17082456, 17693259, 20304795, 25562883
Functional class of membraneless organelle: biomolecular filter/selectivity barrier
Binding partners (at biological protein concentrations)
N/A
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required.
Molecular interaction types contributing to LLPS
gelation (PMID:17082456) π-π (pi-pi) interactions (PMID:17082456)
Determinants of phase separation and droplet properties
1) protein concentration of Nsp1
Membrane cluster No
Partner-dependent No
RNA-dependent No
PTM required No
Domain-motif interactions No
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
The FG repeats of NUPs can form an elastic hydrogel (morphology) in aqueous solution in vitro as demonstrated by gelling of 26 mg/ml wild-type fsFG-repeat domain from Nsp1 (400 μM). The F→S mutated repeat domain showed no signs of gelling and remained liquid in aqueous solution even at high concentrations (morphology). Thus, inter-repeat contacts between phenylalanines caused the gelling of the wild-type Nsp1 fsFG-repeat domain. FRAP measurement indicated that the fluorescently labeled wild-type Nsp1 fsFG-repeat domain was nearly immobile within the wild-type fsFG-repeat hydrogel. A fluorescently labeled F→S mutated repeat domain showed no interaction and diffused freely within the wild-type gel. The F→Y mutated Nsp1-repeat domains formed a homotypic hydrogel, but failed to bind NTRs (physical interaction), probably because the additional OH group at the phenyl ring cannot be accommodated into the FXFG binding pockets of the NTRs. Remarkably, nsp1 with F→Y mutated repeats fully complemented the removal of NSP1 in a wild-type background in vivo, while Nsp1 F→S could not (other change in phenotype/functional readout). These results indicate that not only the NTR binding but inter-repeat contacts and, hence, hydrogel formation is also required for NPC function and viability (PMID:17082456). The permeability properties of NUP FG repeat hydrogels have been studied using the N-terminal region the Nsp1 protein. Permeability properties of saturated FG-hydrogels resembled those of NPCs, but unsaturated FG-hydrogels did not (morphology, other change in phenotype/functional readout). The saturated FG-hydrogel (change in protein concentration) became an efficient barrier that firmly excluded the acRedStar protein (inert probe macromolecule). The specific NTR probe, importin β rapidly dissolved within the gel, reaching a partition coefficient of >100. The FG-hydrogel thus behaved as a selective phase constituting an excellent solvent for importin β (PMID:17693259). The N-terminal segment of Nsp1p (residues 2-277) alone forms more strong hydrogels (morphology), presence of the charged C-terminal FxFG repeats of Nsp1p (residues 274-601) apparently modulate the gel strength such that NTRs can move ≈3-fold faster through the gel in vitro. Therefore, in contrast to pathological amyloids, inter-FG repeat contacts do not result in irreversible aggregates (PMID:20304795). The regular and highly charged part of Nsp1 FG domain (residues 274–601) failed to form particles (particle size and count by microscopy) at 10 µM domain concentration on its own (PMID:25562883).
Experimental observations supporting the liquid material state of the condensate
other: FG NUPs form hydrogels through phase separation (PMID:17082456, PMID:25562883)